Small molecules inhibit the interaction of Nrf2 and the Keap1 Kelch domain through a non-covalent mechanism.

Keap1 binds to the Nrf2 transcription factor to promote its degradation, resulting in the loss of gene products that protect against oxidative stress. While cell-active small molecules have been identified that modify cysteines in Keap1 and effect the Nrf2 dependent pathway, few act through a non-covalent mechanism. We have identified and characterized several small molecule compounds that specifically bind to the Keap1 Kelch-DC domain as measured by NMR, native mass spectrometry and X-ray crystallography. One compound upregulates Nrf2 response genes measured by a luciferase cell reporter assay. The non-covalent inhibition strategy presents a reasonable course of action to avoid toxic side-effects due to non-specific cysteine modification.

Fumarates promote cytoprotection of central nervous system cells against oxidative stress via the nuclear factor (erythroid-derived 2)-like 2 pathway.

Oxidative stress is central to the pathology of several neurodegenerative diseases, including multiple sclerosis, and therapeutics designed to enhance antioxidant potential could have clinical value. The objective of this study was to characterize the potential direct neuroprotective effects of dimethyl fumarate (DMF) and its primary metabolite monomethyl fumarate (MMF) on cellular resistance to oxidative damage in primary cultures of central nervous system (CNS) cells and further explore the dependence and function of the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathway in this process. Treatment of animals or primary cultures of CNS cells with DMF or MMF resulted in increased nuclear levels of active Nrf2, with subsequent up-regulation of canonical antioxidant target genes. DMF-dependent up-regulation of antioxidant genes in vivo was lost in mice lacking Nrf2 [Nrf2(-/-)]. DMF or MMF treatment increased cellular redox potential, glutathione, ATP levels, and mitochondrial membrane potential in a concentration-dependent manner. Treating astrocytes or neurons with DMF or MMF also significantly improved cell viability after toxic oxidative challenge in a concentration-dependent manner. This effect on viability was lost in cells that had eliminated or reduced Nrf2. These data suggest that DMF and MMF are cytoprotective for neurons and astrocytes against oxidative stress-induced cellular injury and loss, potentially via up-regulation of an Nrf2-dependent antioxidant response. These data also suggest DMF and MMF may function through improving mitochondrial function. The clinical utility of DMF in multiple sclerosis is being explored through phase III trials with BG-12, which is an oral therapeutic containing DMF as the active ingredient.

Fumaric acid esters exert neuroprotective effects in neuroinflammation via activation of the Nrf2 antioxidant pathway.

Inflammation and oxidative stress are thought to promote tissue damage in multiple sclerosis. Thus, novel therapeutics enhancing cellular resistance to free radicals could prove useful for multiple sclerosis treatment. BG00012 is an oral formulation of dimethylfumarate. In a phase II multiple sclerosis trial, BG00012 demonstrated beneficial effects on relapse rate and magnetic resonance imaging markers indicative of inflammation as well as axonal destruction. First we have studied effects of dimethylfumarate on the disease course, central nervous system, tissue integrity and the molecular mechanism of action in an animal model of chronic multiple sclerosis: myelin oligodendrocyte glycoprotein induced experimental autoimmune encephalomyelitis in C57BL/6 mice. In the chronic phase of experimental autoimmune encephalomyelitis, preventive or therapeutic application of dimethylfumarate ameliorated the disease course and improved preservation of myelin, axons and neurons. In vitro, the application of fumarates increased murine neuronal survival and protected human or rodent astrocytes against oxidative stress. Application of dimethylfumarate led to stabilization of the transcription factor nuclear factor (erythroid-derived 2)-related factor 2, activation of nuclear factor (erythroid-derived 2)-related factor 2-dependent transcriptional activity and accumulation of NADP(H) quinoline oxidoreductase-1 as a prototypical target gene. Furthermore, the immediate metabolite of dimethylfumarate, monomethylfumarate, leads to direct modification of the inhibitor of nuclear factor (erythroid-derived 2)-related factor 2, Kelch-like ECH-associated protein 1, at cysteine residue 151. In turn, increased levels of nuclear factor (erythroid-derived 2)-related factor 2 and reduced protein nitrosylation were detected in the central nervous sytem of dimethylfumarate-treated mice. Nuclear factor (erythroid-derived 2)-related factor 2 was also upregulated in the spinal cord of autopsy specimens from untreated patients with multiple sclerosis. In dimethylfumarate-treated mice suffering from experimental autoimmune encephalomyelitis, increased immunoreactivity for nuclear factor (erythroid-derived 2)-related factor 2 was detected by confocal microscopy in neurons of the motor cortex and the brainstem as well as in oligodendrocytes and astrocytes. In mice deficient for nuclear factor (erythroid-derived 2)-related factor 2 on the same genetic background, the dimethylfumarate mediated beneficial effects on clinical course, axon preservation and astrocyte activation were almost completely abolished thus proving the functional relevance of this transcription factor for the neuroprotective mechanism of action. We conclude that the ability of dimethylfumarate to activate nuclear factor (erythroid-derived 2)-related factor 2 may offer a novel cytoprotective modality that further augments the natural antioxidant responses in multiple sclerosis tissue and is not yet targeted by other multiple sclerosis therapies.

TRAF3 controls activation of the canonical and alternative NFkappaB by the lymphotoxin beta receptor.

Components of lymphotoxin beta receptor (LTBR)-associated signaling complexes, including TRAF2, TRAF3, NIK, IKK1, and IKK2 have been shown to participate in the coupling of LTBR to NFkappaB. Here, we report that TRAF3 functions as a negative regulator of LTBR signaling via both canonical and non-canonical NFkappaB pathways by two distinct mechanisms. Analysis of NFkappaB signaling in cell lines with functionally intact NFkappaB pathway but lacking LTBR-mediated induction of NFkappaB target genes revealed an inverse association of cellular TRAF3 levels with LTBR-specific defect in canonical NFkappaB activation. Increased expression of TRAF3 correlated with its increased recruitment to LTBR-induced signaling complexes, decreased recruitment of TRAF2, and attenuated phosphorylation of IkappaB alpha and RelA. In contrast, activation of NFkappaB by TNF did not depend on TRAF3 levels. siRNA-mediated depletion of TRAF3 promoted recruitment of TRAF2 and IKK1 to activated LTBR, enabling LTBR-inducible canonical NFkappaB signaling and NFkappaB target gene expression. TRAF3 knock-down also increased mRNA and protein expression of several non-canonical NFkappaB components, including NFkappaB2/p100, RelB, and NIK, accompanied by processing of NFkappaB2/p100 into p52. These effects of TRAF3 depletion did not require LTBR signaling and were consistent with autonomous activation of the non-canonical NFkappaB pathway. Our data illustrate the function of TRAF3 as a dual-mode repressor of LTBR signaling that controls activation of canonical NFkappaB, and de-repression of the intrinsic activity of non-canonical NFkappaB. Modulation of cellular TRAF3 levels may thus contribute to regulation of NFkappaB-dependent gene expression by LTBR by affecting the balance of LTBR-dependent activation of canonical and non-canonical NFkappaB pathways.

Inhibition of integrin alpha(v)beta6, an activator of latent transforming growth factor-beta, prevents radiation-induced lung fibrosis.

RATIONALE: In experimental models, lung fibrosis is dependent on transforming growth factor (TGF)-beta signaling. TGF-beta is secreted in a latent complex with its propeptide, and TGF-beta activators release TGF-beta from this complex. Because the integrin alpha(v)beta6 is a major TGF-beta activator in the lung, inhibition of alpha(v)beta6-mediated TGF-beta activation is a logical strategy to treat lung fibrosis. OBJECTIVES: To determine, by genetic and pharmacologic approaches, whether murine radiation-induced lung fibrosis is dependent on alpha(v)beta6. METHODS: Wild-type mice, alpha(v)beta6-deficient (Itgb6-/-) mice, and mice heterozygous for a Tgfb1 mutation that eliminates integrin-mediated activation (Tgfb1(+/RGE)) were exposed to 14 Gy thoracic radiation. Some mice were treated with an anti-alpha(v)beta6 monoclonal antibody or a soluble TGF-beta receptor fusion protein. Alpha(v)beta6 expression was determined by immunohistochemistry. Fibrosis, inflammation, and gene expression patterns were assessed 20-32 weeks postirradiation. MEASUREMENTS AND MAIN RESULTS: Beta6 integrin expression increased within the alveolar epithelium 18 weeks postirradiation, just before onset of fibrosis. Itgb6-/- mice were completely protected from fibrosis, but not from late radiation-induced mortality. Anti-alpha(v)beta6 therapy (1-10 mg/kg/wk) prevented fibrosis, but only higher doses (6-10 mg/kg/wk) caused lung inflammation similar to that in Itgb6-/- mice. Tgfb1-haploinsufficient mice were also protected from fibrosis. CONCLUSIONS: Alpha(v)beta6-mediated TGF-beta activation is required for radiation-induced lung fibrosis. Together with previous data, our results demonstrate a robust requirement for alpha(v)beta6 in distinct fibrosis models. Inhibition of alphavbeta6-mediated TGF-beta activation is a promising new approach for antifibrosis therapy.

Partial inhibition of integrin alpha(v)beta6 prevents pulmonary fibrosis without exacerbating inflammation.

RATIONALE: Transforming growth factor (TGF)-beta has a central role in driving many of the pathological processes that characterize pulmonary fibrosis. Inhibition of the integrin alpha(v)beta6, a key activator of TGF-beta in lung, is an attractive therapeutic strategy, as it may be possible to inhibit TGF-beta at sites of alpha(v)beta6 up-regulation without affecting other homeostatic roles of TGF-beta. OBJECTIVES: To analyze the expression of alpha(v)beta6 in human pulmonary fibrosis, and to functionally test the efficacy of therapeutic inhibition of alpha(v)beta6-mediated TGF-beta activation in murine bleomycin-induced pulmonary fibrosis. METHODS: Lung biopsies from patients with a diagnosis of systemic sclerosis or idiopathic pulmonary fibrosis were stained for alpha(v)beta6 expression. A range of concentrations of a monoclonal antibody that blocks alpha(v)beta6-mediated TGF-beta activation was evaluated in murine bleomycin-induced lung fibrosis. MEASUREMENTS AND MAIN RESULTS: Alpha(v)beta6 is overexpressed in human lung fibrosis within pneumocytes lining the alveolar ducts and alveoli. In the bleomycin model, alpha(v)beta6 antibody was effective in blocking pulmonary fibrosis. At high doses, there was increased expression of markers of inflammation and macrophage activation, consistent with the effects of TGF-beta inhibition in the lung. Low doses of antibody attenuated collagen expression without increasing alveolar inflammatory cell populations or macrophage activation markers. CONCLUSIONS: Partial inhibition of TGF-beta using alpha(v)beta6 integrin antibodies is effective in blocking murine pulmonary fibrosis without exacerbating inflammation. In addition, the elevated expression of alpha(v)beta6, an activator of the fibrogenic cytokine, TGF-beta, in human pulmonary fibrosis suggests that alpha(v)beta6 monoclonal antibodies could represent a promising new therapeutic strategy for treating pulmonary fibrosis.

Pharmacodynamics of Dimethyl Fumarate Are Tissue Specific and Involve NRF2-Dependent and -Independent Mechanisms.

AIMS: Gastro-resistant dimethyl fumarate (DMF) is an oral therapeutic indicated for the treatment of relapsing multiple sclerosis. Recent data suggest that a primary pharmacodynamic response to DMF treatment is activation of the nuclear factor (erythroid-derived 2)-like 2 (NRF2) pathway; however, the gene targets modulated downstream of NRF2 that contribute to DMF-dependent effects are poorly understood. RESULTS: Using wild-type and NRF2 knockout mice, we characterized DMF transcriptional responses throughout the brain and periphery to understand DMF effects in vivo and to explore the necessity of NRF2 in this process. Our findings identified tissue-specific expression of NRF2 target genes as well as NRF2-dependent and -independent gene regulation after DMF administration. Furthermore, using gene ontology, we identified common biological pathways that may be regulated by DMF and contribute to in vivo functional effects. INNOVATION: Together, these data suggest that DMF modulates transcription through multiple pathways, which has implications for the cytoprotective, immunomodulatory, and clinical properties of DMF. CONCLUSION: These findings provide further understanding of the DMF mechanism of action and propose potential therapeutic targets that warrant further investigation for treating neurodegenerative diseases. Antioxid. Redox Signal. 24, 1058-1071.

Pristane-Accelerated Autoimmune Disease in (SWR X NZB) F1 Mice Leads to Prominent Tubulointerstitial Inflammation and Human Lupus Nephritis-Like Fibrosis.

Mouse models lupus nephritis (LN) have provided important insights into disease pathogenesis, although none have been able to recapitulate all features of the human disease. Using comprehensive longitudinal analyses, we characterized a novel accelerated mouse model of lupus using pristane treatment in SNF1 (SWR X NZB F1) lupus prone mice (pristane-SNF1 mice). Pristane treatment in SNF1 mice accelerated the onset and progression of proteinuria, autoantibody production, immune complex deposition and development of renal lesions. At week 14, the pristane-SNF1 model recapitulated kidney disease parameters and molecular signatures seen in spontaneous disease in 36 week-old SNF1 mice and in a traditional IFNα-accelerated NZB X NZW F1 (BWF1) model. Blood transcriptome analysis revealed interferon, plasma cell, neutrophil, T-cell and protein synthesis signatures in the pristane-SNF1 model, all known to be present in the human disease. The pristane-SNF1 model appears to be particularly useful for preclinical research, robustly exhibiting many characteristics reminiscent of human disease. These include i) a stronger upregulation of the cytosolic nucleic acid sensing pathway, which is thought to be key component of the pathogenesis of the human disease, and ii) more prominent kidney interstitial inflammation and fibrosis, which have been both associated with poor prognosis in human LN. To our knowledge, this is the only accelerated model of LN that exhibits a robust tubulointerstitial inflammatory and fibrosis response. Taken together our data show that the pristane-SNF1 model is a novel accelerated model of LN with key features similar to human disease.

Alphav beta6 integrin regulates renal fibrosis and inflammation in Alport mouse.

The transforming growth factor (TGF)-beta-inducible integrin alpha v beta6 is preferentially expressed at sites of epithelial remodeling and has been shown to bind and activate latent precursor TGF-beta. Herein, we show that alpha v beta6 is overexpressed in human kidney epithelium in membranous glomerulonephritis, diabetes mellitus, IgA nephropathy, Goodpasture's syndrome, and Alport syndrome renal epithelium. To assess the potential regulatory role of alpha v beta6 in renal disease, we studied the effects of function-blocking alpha v beta6 monoclonal antibodies (mAbs) and genetic ablation of the beta6 subunit on kidney fibrosis in Col4A3-/- mice, a mouse model of Alport syndrome. Expression of alpha v beta6 in Alport mouse kidneys was observed primarily in cortical tubular epithelial cells and in correlation with the progression of fibrosis. Treatment with alpha v beta6-blocking mAbs inhibited accumulation of activated fibroblasts and deposition of interstitial collagen matrix. Similar inhibition of renal fibrosis was observed in beta6-deficient Alport mice. Transcript profiling of kidney tissues showed that alpha v beta6-blocking mAbs significantly inhibited disease-associated changes in expression of fibrotic and inflammatory mediators. Similar patterns of transcript modulation were produced with recombinant soluble TGF-beta RII treatment, suggesting shared regulatory functions of alpha v beta6 and TGF-beta. These findings demonstrate that alpha v beta6 can contribute to the regulation of renal fibrosis and suggest this integrin as a potential therapeutic target.

Targeting the lymphotoxin-beta receptor with agonist antibodies as a potential cancer therapy.

The lymphotoxin-beta receptor (LT beta R) is a tumor necrosis factor receptor family member critical for the development and maintenance of various lymphoid microenvironments. Herein, we show that agonistic anti-LT beta R monoclonal antibody (mAb) CBE11 inhibited tumor growth in xenograft models and potentiated tumor responses to chemotherapeutic agents. In a syngeneic colon carcinoma tumor model, treatment of the tumor-bearing mice with an agonistic antibody against murine LT beta R caused increased lymphocyte infiltration and necrosis of the tumor. A pattern of differential gene expression predictive of cellular and xenograft response to LT beta R activation was identified in a panel of colon carcinoma cell lines and when applied to a panel of clinical colorectal tumor samples indicated 35% likelihood a tumor response to CBE11. Consistent with this estimate, CBE11 decreased tumor size and/or improved long-term animal survival with two of six independent orthotopic xenografts prepared from surgical colorectal carcinoma samples. Targeting of LT beta R with agonistic mAbs offers a novel approach to the treatment of colorectal and potentially other types of cancers.

Topology of gene expression networks as revealed by data mining and modeling.

MOTIVATION: Interpretation of high-throughput gene expression profiling requires a knowledge of the design principles underlying the networks that sustain cellular machinery. Recently a novel approach based on the study of network topologies has been proposed. This methodology has proven to be useful for the analysis of a variety of biological systems, including metabolic networks, networks of protein-protein interactions, and gene networks that can be derived from gene expression data. In the present paper, we focus on several important issues related to the topology of gene expression networks that have not yet been fully studied. RESULTS: The networks derived from gene expression profiles for both time series experiments in yeast and perturbation experiments in cell lines are studied. We demonstrate that independent from the experimental organism (yeast versus cell lines) and the type of experiment (time courses versus perturbations) the extracted networks have similar topological characteristics suggesting together with the results of other common principles of the structural organization of biological networks. A novel computational model of network growth that reproduces the basic design principles of the observed networks is presented. Advantage of the model is that it provides a general mechanism to generate networks with different types of topology by a variation of a few parameters. We investigate the robustness of the network structure to random damages and to deliberate removal of the most important parts of the system and show a surprising tolerance of gene expression networks to both kinds of disturbance.

Dual role for TWEAK in angiogenic regulation.

Angiogenic regulators modulate endothelial cell functions, including proliferation, migration, secretion, and adhesion, through their action on endothelial cells or other cell types. TWEAK, a novel member of the tumor necrosis factor family, appears to be a pro-angiogenic agent on the basis of previous studies demonstrating its ability to induce interleukin-8 production by epithelial tumor lines, stimulate proliferation of human vascular cell types and neovascularization in rat corneas. Here, we further characterized the angiogenic potential of TWEAK, revealing a dual role for TWEAK as an angiogenic regulator. We demonstrate that TWEAK is a potent inducer of endothelial cell survival and cooperates with basic fibroblast growth factor to induce the proliferation and migration of human endothelial cells and morphogenesis of capillary lumens. In contrast, TWEAK antagonizes the morphogenic response of endothelial cells to vascular endothelial growth factor (VEGF) without inhibiting VEGF-induced survival or proliferation. Thus, our observations suggest that TWEAK may differentially regulate microvascular growth, remodeling and/or maintenance in vivo, depending upon the angiogenic context.